Cofactor analogue-induced chemical reactivation of endonucleaseactivity in a DNA cleavage/methylation deficient TspGWI N473Avariant in the NPPY motif

Agnieszka Zylicz-Stachula • Joanna Je _zewska-Frackowiak •

Piotr M. Skowron

Received: 18 February 2013 / Accepted: 4 January 2014

� The Author(s) 2014. This article is published with open access at Springerlink.com

Abstract We reported previously that TspGWI, a prototype

enzyme of a new Thermus sp. family of restriction endonu-

cleases-methyltransferases (REases-MTases), undergoes the

novel phenomenon of sinefungin (SIN)-caused specificity

transition. Here we investigated mutant TspGWI N473A,

containing a single amino acid (aa) substitution in the NPPY

motif of the MTase. Even though the aa substitution is located

within the MTase polypeptide segment, DNA cleavage and

modification are almost completely abolished, indicating that

the REase and MTase are intertwined. Remarkably, the

TspGWI N473A REase functionality can be completely

reconstituted by the addition of SIN. We hypothesize that SIN

binds specifically to the enzyme and restores the DNA

cleavage-competent protein tertiary structure. This indicates

the significant role of allosteric effectors in DNA cleavage in

Thermus sp. enzymes. This is the first case of REase mutation

suppression by an S-adenosylmethionine (SAM) cofactor

analogue. Moreover, the TspGWI N473A clone strongly

affects E. coli division control, acting as a ‘selfish gene’. The

mutant lacks the competing MTase activity and therefore

might be useful for applications in DNA manipulation. Here

we present a case study of a novel strategy for REase activity/

specificity alteration by a single aa substitution, based on the

bioinformatic analysis of active motif locations, combining

(a) aa sequence engineering (b) the alteration of protein

enzymatic properties, and (c) the use of cofactor–analogue

cleavage reconstitution and stimulation.

Keywords Endonuclease-methyltransferase � Thermus

sp. enzyme � Enzymatic reaction cofactor � Cofactor

analogue � Sinefungin � S-adenosylmethione � Mutant

activation � Specificity change

Introduction

With the advent of bioinformatics, research into the

restriction-modification (RM) of DNA received a strong

impulse, both to further evaluate basic research aspects and

develop new tools for genetic engineering. After reaching a

relative plateau over 10 years ago in the search for new

prototype specificities using the classic biochemical

method, rapid bacterial genome and metagenomic

sequencing coupled with homology, evolutionary and

advanced active motif software predictions has currently

opened up a new chapter in the efficient search for gene

candidates for new RM as well as structure–function and

DNA sequence-protein folding analysis [1–5]. Sequence-

structure–function bioinformatics studies provide data for

the controlled sequence-specific protein specificity/activity

determination of functionally critical aa as well as rational

protein engineering. In contrast to the majority of proteins,

a significant sequence similarity between REases is extre-

mely rare, even when isoschizomers are compared. Recent

advances relating to the construction of a homology model

MwoI with DNA have pinpointed functionally important aa

that have subsequently been targeted by mutagenesis,

resulting in changes in enzyme specificity [6]. With the

discovery of new genes and enzymes, numerous atypical

A. Zylicz-Stachula (&) � J. Je _zewska-Frackowiak �P. M. Skowron (&)

Department of Molecular Biotechnology, Institute for

Environmental and Human Health Protection, Faculty of

Chemistry, University of Gdansk, Wita Stwosza 63,

80-952 Gdansk, Poland

e-mail: a.zylicz-stachula@ug.edu.pl

P. M. Skowron

e-mail: pmars44@gmail.com; piotr.skowron@ug.edu.pl

123

Mol Biol Rep

DOI 10.1007/s11033-014-3085-x

REases have been discovered in recent years, which depart

from established paradigms, such as the radically different

magnesium-independent BfiI REase. This evolved by an

entirely different route than other REases, emerging as a

fusion protein between a Mg2?-independent non-specific

nuclease and a B3-like DNA-binding domain from plant

transcription regulatory protein [7]. Interestingly, bioin-

formatics studies have shown that a BfiI homologue exists

in Mesorhizobium sp. bacteria BNC, which is a symbiont of

a plant capable of growing in an EDTA-rich environment

[7]. Another natural REase – CviJI of unusual eukaryotic

origin, coded by IL-3A virus infected Chlorella algae—is

an adenine nucleotide-stimulated enzyme (a feature not

found among other natural REases) that cleaves a 2–3 bp

cognate site. It is therefore the most frequently recognized

DNA REase among the over 300 prototypes known [8–10].

We have constructed two chemically modified, frequently

cleaving artificial REases, namely TspGWI/SIN, an alter-

native to CviJI, cleaving the 3-bp averaged prototype site

[11], and TaqII/SIN/DMSO, cleaving the 2.9-bp averaged

prototype site [12, 13].

Both TspGWI and TaqII belong to the Thermus sp.

enzyme family—another atypical group of REases, which

we have defined [11–17, 19]. The existing members of the

family include six related thermostable enzymes: TspGWI

[ACGGA (11/9)] [15, 16], TspDTI [(ATGAA (11/9)] [14,

17], Tth111II/TthHB27I [(CAARCA (11/9)] [14, 17, 18],

TsoI [TARCCA (11/9)] [10, 17, 19] and TaqII [(GACCGA

(11/9) or CACCCA (11/9)] [12, 20]. Recent analysis and

literature data have shown the existence of putative or

partially analysed members (or genes) of the Thermus sp.

family, originating from evolutionarily distant mesophilic

bacteria [10, 19]. This may indicate two possible routes by

which the family evolved: (i) divergent evolution following

horizontal interspecies transfer from a common ancestor of

the family, or (ii) the formation of an enzyme pro-proto-

type even earlier in a single bacteria species and further

proliferating its specificity and other features as the host

evolves and gives origin to a new bacterial species/strains.

In this paper we describe further studies on the TspGWI

bifunctional REase, showing a novel type of mutation

suppression induced by a cofactor analogue, where the

NPPY methylation catalytic motif is converted to a non-

functional APPY segment, while the restriction activity

becomes reactivated in vitro.

Materials and methods

Bacterial strains, plasmids, media and reagents

A wild-type (wt), recombinant TspGWI protein expression

plasmid (pRZ-TspGWI) was constructed previously [16].

Site-directed mutagenesis within the tspGWIRM gene to

make pRZ-TspGWI APPY was described previously [16].

The procedures of recombinant wt TspGWI clone cultur-

ing, induction and protein purification were amended for

the properties of the TspGWI N473A variant (NPPY motif

to APPY transition) in order to ensure that thermostable

protein folding conditions were maintained. Marathon

DNA Polymerase was from A&A Biotechnology (Gdansk,

Poland), and T7 bacteriophage DNA, plasmids pBR322

were from Vivantis Technologies (Shah Alam, Malay-

sia).The PCR primer synthesis was performed at Genomed

(Warsaw, Poland). All other reagents were purchased from

Sigma-Aldrich (St Louis, MO, USA). 100 bp DNA and

1 kb DNA markers were from Fermentas (Thermo Fisher

Scientific, MA, USA).

Expression of the tspGWIRM (N473A) gene

under control of the PR promoter in E. coli

and purification of the TspGWI N473A enzyme

Escherichia coli DH11S [pRZ-TspGWI APPY] was

expressed in TB medium supplemented with chloram-

phenicol (40 lg/ml) and maltose (0.5 %) at 30 �C with

vigorous aeration, followed by PR promoter induction as a

result of a temperature shift to 42 �C. Simultaneously,

expression of the wt tspGWIRM clone was performed,

under the same conditions, for growth and induction con-

trol purposes. Expression of tspGWIRM (N473A) in E. coli

DH11S [pRZ-TspGWI APPY] was initiated with bacterial

inoculum flushed from a Petri dish into 500 ml of medium.

The culture was grown with vigorous aeration until OD600

reached 0.6, and the temperature shift was performed by

the addition of 500 ml of medium pre-warmed to 60 �C.

The culture was further supplemented with chlorampheni-

col. Uninduced control and induced cells were subjected to

10 % SDS-PAGE, and gels were analysed for the appear-

ance of the expected polypeptide size of app. 120 kDa and

for endonucleolytic activity in crude lysates (recombinant

wt TspGWI only). The recombinant wt TspGWI and

TspGWI N473A proteins concentrations were determined

utilizing Coomassie Brillant Blue stained protein band

densitometric analysis, with the use of BSA serial dilutions

calibration curve. The concentration values were obtained

from a linear standard reflective scan mode with back-

ground correction in UN-SCAN IT GEL for Windows 6.1

data software (v. 6.1, Gel Analysing and Graph Digitizing

Software, Silk Scientific Corporation, Orem, UT, USA).

Additionally, 4 h after induction, samples for microscopic

analysis were taken from both recombinant wt TspGWI

and TspGWI N473A mutant cultures. Bacteria were stained

following the standard methylene blue positive staining

protocol [21]. Slide glasses preparations were observed and

photographed with immerse objective lenses of 1009

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magnification, under an Olympus CX21FS1 light micro-

scope with a total obtained magnification of 12009

(Fig. 1). The culture growth was continued for 14 h at

42 �C; both variants of TspGWI were accumulating

slowly, becoming detectable after only 2 h. The purifica-

tion scheme varied from the one we described previously

for the native (Thermus sp. GW-purified) and recombinant

wt TspGWI (E. coli-purified) enzymes [16], and included

the following stages:

1. Polyethyleneimine (PEI) removal of nucleic acids

from a sonicated and centrifuged bacterial extract,

obtained by suspension of 16 g of cells in 4 volumes of

buffer A [20 mM Tris–HCl (pH 8.0 at 25 �C), 0.5 mM

EDTA, 50 mM NaCl, 5 % glycerol, 10 mM 2-mercap-

toethanol (ßMe), 1 mM PMSF, 1 mg/ml lysozyme],

addition of NaCl to 400 mM concentration, and the

gradual addition of PEI to 0.4 %. Following 30 min. of

stirring, the sample was centrifuged and the superna-

tant subjected to ammonium sulphate (AmS)

fractionation.

2. AmS fractionation was conducted in two phases. In the

first step, 35 % saturation (at 4 �C, 0.208 g/ml), con-

taminating proteins were removed. In the second stage,

50 % saturation was applied (additional 0.094 g/ml),

the suspension stirred overnight, centrifuged, dis-

solved in buffer B and dialysed against buffer B

[20 mM K/PO4 (pH 8.0 at 25 �C), 0.5 mM EDTA,

50 mM NaCl, 0.02 % Triton X-100, 0.02 % Tween

20, 5 % glycerol, 10 mM ßMe,1 mM PMSF].

3. Phosphocellulose chromatography was conducted in

buffer B and the proteins eluted with the following

NaCl steps in buffer B (mM): 100, 200, 300, 400, 500

and 1 M. TspGWI variants were dialysed against

buffer C [20 mM Tris–HCl (pH 8.0 at 25 �C), 0.5 mM

EDTA, 30 mM NaCl, 0.01 % Triton X-100, 0.01 %

Tween 20, 5 % glycerol, 10 mM ßMe, 0.1 mM

PMSF]. TspGWI variants eluted at 200–300 mM

NaCl.

4. DEAE-Sephadex chromatography using buffer C

employed NaCl steps in buffer C (mM): 100, 150,

200, 250, 300, 350, 400, 450 and 500. TspGWI

variants eluted at 150–200 mM NaCl. The DEAE-

Sephadex chromatography was repeated twice. Pooled

column fractions containing the enzyme were dialysed

against buffer C between repeated procedures and

finally against buffer D [20 mM K/PO4 (pH 7.0 at

25 �C), 100 mM NaCl, 0.01 % Triton X-100, 5 %

glycerol, 10 mM ßMe, 0.1 mM PMSF].

5. Hydroxyapatite chromatography was used to adsorb

TspGWI proteins, the column was flushed with buffer

D, and the following K/PO4 buffer steps were used for

elution (mM): 40, 80, 120, 160, 200, 240 and 280.

TspGWI variants eluted at 150–200 mM K/PO4. Final

preparations were dialysed against storage buffer P

[20 mM Tris–HCl (pH 8.3 at 25 �C), 0.1 mM EDTA,

25 mM KCl, 40 mM AmS, 0.05 % Tween 20, 0.5 mM

DTT, 50 % glycerol].

The purity of both enzyme preparations was estimated

on a 10 % SDS-PAGE gel. Loaded samples contained 2 lg

of wt or mutant TspGWI proteins (Fig. 2).

REase activity in DNA cleavage assay

Standardized conditions for DNA cleavage were used, both

in the presence and absence of 50 lM SAM (cofactor) or

SIN (cofactor analogue), in an optimal reaction buffer

T [50 mM Tris–HCl (pH 7.2 at 65 �C), 10 mM

MgCl2,10 mM DTT] [15]. The reaction volume of 50 ll

contained either 300 ng bacteriophage T7 DNA or 300 ng

custom PCR substrate. All experiments utilized recombi-

nant wt TspGWI or TspGWI N473A variant protein. Under

those conditions both SAM and SIN exhibited sufficient

Fig. 1 Microscope imaging of methylene blue stained E. coli cells

harbouring wt tspGWIRM or tspGWIRM N473A gene. Samples of the

bacterial culture were taken both from E. coli cells expressing wt

TspGWI and TspGWI N473A variant, 4 h after promoter PR

transcription induction. After standard methylene blue positive

staining, slide glasses preparations were observed under Olympus

CX21FS1 light microscope with total 91200 magnification

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stability (not shown). The titration experiments were car-

ried out as a series of two-fold dilutions, starting from

500 ng (4.16 pmol) down to 3.91 ng (33 fmol) of wt

TspGWI protein or TspGWI N473A mutant (Figs. 3, 4).

SIN titration (Fig. 5) or cleavage specificity determination

(Fig. 6) were performed in the presence of 4.16 pmol of wt

TspGWI or TspGWI N473A. To precisely compare small

differences in digestions extent, the reactions were carried

out at enzyme/time-limiting conditions, thus avoiding

overdigestion conditions. After 1 h at 65 �C the reactions

were immediately stopped by the addition of EDTA, SDS,

proteinase K and the REases were digested for 1 h at

55 �C, then the DNA was ethanol-precipitated. The DNA

precipitate was collected by centrifugation and redissolved

in 10 mM Tris–HCl (pH 8.0 at 25 �C). The custom 390 bp

PCR fragment [11] was constructed for comparative titra-

tions of TspGWI protein variants (Fig. 4). The PCR con-

taining double convergent (?/) canonical sites for

TspGWI was constructed using a pair of primers: 50-CTCG

ACCTGAATGGAAGCCG-30 and 50-GGTGCAGGGCG

CTGACTTCC-30, amplifying a modified DNA segment

from pBR322 plasmid. The TspGWI ‘zero sites’ custom

390 bp PCR variant was constructed using a pair of PCR

mutagenic primers: 50-CTCGACCTGAATGGAAGC

CGGCGGCACCTCGCTGACCGATTCACCACT-30 and

50-GGTGCAGGGCGCTGACTTCCGCGTTTCCAGACT

TTACGAAACACCCAAACCGAAGA-3’. For the pur-

pose of clearer interpretation of the TspGWI N473A mutant

cleavage specificity assay (Fig. 6), the distances from both

50 and 30 PCR ends to the TspGWI recognition sites were

extended in both the ‘double convergent’ and ‘zero sites’

custom substrates, resulting in two, nearly identical 497 bp

PCR fragments, with point bp changes within the TspGWI

recognition sequences. The prolonging primers comple-

mentary to the 390 bp PCR were designated F63-390 and

R75-390, as we described previously [12]. The electro-

phoresis samples were heated at 65 �C for 5 min. in a

loading buffer, containing SDS and EDTA to facilitate

DNA release from complexes with TspGWI protein vari-

ants. The PCR DNA cleavage products were analysed by

15 % polyacrylamide gel electrophoresis in TBE buffer,

while T7 DNA cleavage products were resolved in a 1.3 %

TBE agarose gel. Complete digestion of the 390 bp PCR

product by TspGWI would yield 282 bp, 56 bp and 48 bp

fragments. Complete digestion of the 497 bp PCR product

by TspGWI would yield 282 bp, 116 bp and 95 bp frag-

ments. DNA bands were quantitatively compared by

applying UN-SCAN IT GEL for Windows 6.1 data soft-

ware to a series of photographs taken with different

exposure times.

Results and discussion

The prototype TspGWI-coding gene was the first in a series

of related genes from the Thermus sp. family cloned by our

group [16], and was originally found in the very same ther-

mal water sample as another prototype member of the fam-

ily—TspDTI [14, 17]. Bioinformatics analysis of the

Thermus sp. family RM genes coupled with experimental

confirmation by site-directed mutagenesis [16] defined dis-

tinct functional regions, fused within a single polypeptide:

Type I REases-like domains arranged in tandem. Conspic-

uous are the central HsdM-like module (helical domain),

conserved MTase domain and N-terminal nuclease domain,

similar to the corresponding domains in HsdR subunits. Both

the ATP-dependent translocase module of the HsdR subunit

and the supplementary domains involved in subunit–subunit

interactions in Type I systems are missing. This indicates that

structurally and functionally, the Thermus sp. enzyme pro-

tomers correspond to the streamlined ‘half’ of a Type I

enzyme [16]. The structural and functional domain

arrangement of TspGWI and other Thermus sp. family of

REases was shown in our previous publications [16, 17].

With the use of bioinformatics analysis and preliminary

site-directed mutagenesis studies we designed and con-

structed mutants in TspGWI polypeptide domain motifs,

crucial to REase and MTase activities [16]. These included

REase catalytic motif PD-EXK, the SAM binding motif

DPAVGTG and the typical methylation catalytic motif

NPPY. Somewhat surprisingly, further cloning,

Fig. 2 SDS-PAGE analysis of purified wt recombinant TspGWI and

TspGWI N473A proteins. Purified protein preparations containing

2 lg of wt recombinant TspGWI or TspGWI N473A were electro-

phoresed in 10 % SDS-PAGE. Lane M, protein marker (GE

Healthcare); lane 1, purified protein preparation containing 2 lg of

wt recombinant TspGWI REase; lane 2, purified protein preparation

containing 2 lg of TspGWI N473A variant. Both TspGWI variants are

indicated with an arrow

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biochemical and bioinformatics studies revealed that both

TspGWI and TspDTI, though belonging to the Thermus sp.

family, are more remotely related to each other than to the

other members found in distant hot spring/thermal water

locations (separated by thousands of kilometres and/or

ocean). TspGWI is similar to TaqII and TspDTI resembles

Tth111II, so the family is divided into two sub-families:

TspGWI-like [16] and TspDTI-like [17]. The differences

relate not only to the general aa sequence similarity, but

also to particularly active motifs. Enzymes from the

TspGWI-subfamily—TspGWI, TaqII and the hitherto

uncharacterized RpaI, originating from mesophilic bacteria

[10, 19]—have typical REase catalytic motifs PD-(D/E)XK

[16, 22], methylation catalytic NPPY and SAM binding

motifs (DPA(V/M)GTG [16]. On the other hand, the

enzymes from the TspDTI-subfamily—TspDTI, Tth111II/

TthHB27I, TsoI and the so far uncharacterized CchII,

originating from mesophilic bacteria [10, 19]—have

atypical REase catalytic motifs D-EXE (also detected in

typical Type II BamHI REase) [17], a less frequent NPPW

methylation catalytic motif and cysteine?serine containing

SAM binding motifs (D/P)PACGSG [17]. There is also

another remarkable functional difference. We recently

reported a new phenomenon of SIN-mediated specificity

change (‘star affinity‘) in TspGWI from a 5-bp recognition

site to an averaged 3-bp recognition site [11] and in TaqII

from a 5–6-bp recognition site to an averaged 2.9-bp rec-

ognition site [12, 13]. From these studies conducted on wt

proteins, we set up the hypothesis that SIN, being a

chemical analogue of similar structure, is capable of

mimicking SAM in the protein specific binding pocket, but

is not a methyl group donor. Moreover, SIN possesses a

reversed charge distribution as compared to SAM. The

combination of these features may cause subtle confor-

mational and functional changes in Thermus sp. REases’

tertiary structure upon the cofactor analogue binding and

Fig. 3 T7 bacteriophage DNA cleavage patterns of TspGWI protein

variants in the presence or absence of SAM and its analogue SIN.

300 ng of T7 bacteriophage DNA (0.99 pmol TspGWI recognition

sites) was digested for 1 h at 65 �C with consecutive two-fold REase

dilutions, starting from 500 ng (4.16 pmol; 4.2:1 molar ratio of

enzyme to recognition sequence) down to 3.91 ng (33 fmol; 0.03:1

molar ratio of enzyme to recognition sequence) of either TspGWI or

TspGWI N473A variant in buffer T, without SAM/SIN or supple-

mented with 50 lM of the SAM or SIN. The black arrows indicate

the lanes of estimated identical or nearly identical extent of DNA

digestion. The red arrows indicate lanes with a stable partial digestion

pattern, obtained using the minimum sufficient amount of an enzyme.

a Cleavage pattern of recombinant wt TspGWI in the absence of

SAM and SIN. Lanes M, GeneRulerTM 1 kb DNA Ladder (Thermo

Fisher Scientific), selected bands marked; M2 GeneRulerTM 100 bp

DNA Ladder (Thermo Fisher Scientific), selected bands marked; lane

K, untreated T7 bacteriophage DNA; lane 1–8, T7 DNA cleaved with

the wt TspGWI protein, two-fold serial dilutions series. The reaction

products were resolved on 1.3 % agarose gel in TBE buffer and

stained with ethidium bromide (EtBr). b As in Panel a, except that the

reactions were supplemented with 50 lM of SAM. c As in Panel a,

except that the reactions were supplemented with 50 lM of SIN. d As

in Panel a, except that the reactions were carried out with the TspGWI

N473A variant. e As in Panel a, except that the reactions were carried

out with the TspGWI N473A variant and supplemented with 50 lM of

SAM. f As in Panel a, except that the reactions were carried out with

the TspGWI N473A variant and supplemented with 50 lM of SIN

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consequently alter DNA recognition [12, 13]. In contrast,

none of the several TspDTI-like subfamily enzymes tested

in our laboratory exhibited ‘star affinity’, thus this phe-

nomenon is limited hitherto to enzymes possessing PD-(D/

E)XK, NPPY and (DPA(V/M)GTG functional motifs [13,

17].

Investigating TspGWI mutants [16] in more detail, we

found a combination of unusual biochemical features in

one of the mutants: it contained a single aa substitution of

asparagine to alanine residue in position 473 of its MTase

polypeptide region. The TspGWI N473A, with the NPPY

motif converted to APPY, confirmed the results of the

bioinformatics analysis. This mutant had no MTase activity

[16]. We anticipated serious difficulties in or even the

impossibility of cultivating recombinant E. coli with this

mutant clone, owing to the expected residual TspGWI

REase activity, even though we used reduced cultivation

temperatures (28–30 �C) and modified vectors to diminish

thermostable REase activity in vivo. However, despite

being more fragile (prone to lysis), growing more slowly

than their equivalents with the wt TspGWI clone and

exhibiting altered cell morphology, the mutant-carrying

bacteria were successfully cultured (Fig. 1). Strikingly, the

TspGWI N473A (Fig. 1b) expressing E. coli cells are much

more different morphologically than those expressing wt

TspGWI (Fig. 1a). The TspGWI N473A producing cells are

noticeably longer and the culture population is heteroge-

neous. There is an observed frequent presence of filaments

of variable length, including species which are 5 to over

100 (!)-fold longer than E. coli cells expressing the wt

TspGWI protein (Fig. 1ab). Apparently, this MTase-defi-

cient and active only partially REase mutant, even though

it is a thermostable enzyme (thus less active at induction

temperature of 42 �C), produces enough chromosomal

cleavages, exercising a strong selective pressure on

recombinant E. coli cells, to repair DNA breaks. On the

other hand, the residual REase activity is low enough to be

sub-lethal only, thus allowing cells to survive, by inhibiting

Fig. 4 Comparative titration of TspGWI protein variants on the

390 bp PCR DNA substrate in the presence or absence of SAM or its

analogue SIN. 300 ng of 390 bp custom PCR, containing two

convergent 50-ACGGA-30 recognition sites (2.28 fmol TspGWI

recognition sites), was digested with consecutive two-fold REase

dilutions, starting from 500 ng (4.16 pmol; 1.82:1 molar ratio of

enzyme to recognition sequence) down to 3.91 ng (33 fmol; 0.01:1

molar ratio of enzyme to recognition sequence) of recombinant wt

TspGWI or TspGWI N473A variants in buffer T, supplemented with

50 lM of the SAM or SIN, for 1 h, at 65 �C. a Cleavage pattern of

recombinant wt TspGWI in the absence of SAM and SIN. Lanes M,

GeneRulerTM 100 bp DNA Ladder (Thermo Fisher Scientific),

supplemented with four additional small size DNA marker fragments:

21, 32, 42 and 57 bp (selected bands marked); lane K, untreated PCR

DNA; lane 1–8, PCR DNA cleaved with the recombinant wt TspGWI

protein variant, two-fold serial dilution series. The reaction products

were resolved on 15 % polyacrylamide gel in TBE buffer and stained

with Sybr Green I. b As in Panel a, except that the reactions were

supplemented with 50 lM of SAM. c As in Panel a, except that the

reactions were supplemented with 50 lM of SIN. d As in Panel a,

except that the reactions were carried out with the TspGWI N473A

variant. e As in Panel a, except that the reactions were carried out

with the TspGWI N473A variant and supplemented with 50 lM of

SAM. f As in Panel a, except that the reactions were carried out with

the TspGWI N473A variant and supplemented with 50 lM of SIN

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cells divisions. Conclusion can be drawn that bacterial

chromosome replication and cell division is being slowed

down/stalled as a consequence of the necessity to repair

TspGWI N473A-generated DNA cuts in vivo. As a result,

the active TspGWI N473A clone takes control over

recombinant E. coli division. Thus, it acts as a ‘selfish

gene’. This would corroborate with Kobayashi’s descrip-

tion of RM systems as the ‘minimum form of life’ [25].

Furthermore, the hypothesis describes a ‘selfish RM sys-

tem’, which contains at least two genes: coding for an

MTase and REase. Here we show this ‘minimum form of

life’ pushed to the extreme: just a single TspGWI N473A-

coding gene replaces the ‘toxin-antitoxin’ system (cognate

REase-MTase pair) [24]. In this work the DNA modifying

site-specific ‘antitoxin’ (cognate MTase) is apparently

functionally replaced by non-specific, cellular E. coli DNA

damage repair systems. In accordance with the in vivo

cultivation results, the TspGWI N473A expression experi-

ments revealed low REase activity in vitro in crude

recombinant E. coli extracts, despite the comparable

amounts of both mutant and recombinant wt TspGWI

proteins synthesized in vivo. Evaluating the phenomenon,

we purified the TspGWI N473A [16] ( Fig. 2, see Methods

and Table 1) and compared it with the recombinant wt

TspGWI. Both recombinant enzymes were obtained

essentially homogeneous, with minor contaminating bands

visible only upon electrophoresis gel overloading. These

preparations were further used for all analyses described in

this paper (Fig. 2). While retaining the same general bio-

chemical features, such as molecular size, isoelectric point,

domain organization, pH, salt, magnesium ions require-

ments (not shown), owing to the single aa substitution, the

Fig. 5 Comparative SIN titrations in the presence of wt TspGWI

REase or TspGWI N473A variant protein. 300 ng of T7 bacteriophage

DNA (0.99 pmol restriction sites) was digested with 500 ng

(4.16 pmol; 4.2:1 molar ratio of enzyme to recognition sequence)

of a protein variant TspGWI or TspGWI N473A in buffer T,

supplemented with consecutive two-fold SIN dilutions, starting from

100 lM of the SIN down to 3.75 9 10-3 lM (0.375 nM), for 1 h at

65 �C. The black arrow indicates the first lane with lower than

stoichiometric concentration of SIN. a Wt TspGWI-generated

cleavage pattern in the presence of consecutive two-fold SIN

dilutions. Lanes M, GeneRulerTM 1 kb DNA Ladder (Thermo Fisher

Scientific), selected bands marked; lane K1, untreated T7 bacterio-

phage DNA; lane K2, T7 DNA cleaved with the wt TspGWI protein,

without SIN; lane 1–19, T7 DNA cleaved with the recombinant wt

TspGWI, supplemented with SIN, two-fold dilutions series. The

reaction products were resolved on 1.3 % agarose gel in TBE buffer

and stained with EtBr. b As in Panel a, except that the reactions were

carried out with the TspGWI N473A variant

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REase activity of the TspGWI N473A variant (mutation

within MTase domain) was barely detectable. Less than

0.8 % of the relative DNA cleavage activity was detected,

in comparison to the recombinant wt TspGWI as assayed

on the TspGWI-recognition site rich substrate—T7 DNA

(Table 1; Fig. 3) or no activity was detected as assayed on

the 390 bp PCR product containing two convergent

TspGWI sites (Table 1; Fig. 4). The PCR fragment with

two sites was selected to avoid a negative bias towards

single site substrates, which we reported previously for

TspGWI [11]. This result clearly shows the existence of

intertwined communication and the mutual dependence of

REase and MTase functions in the same polypeptide. In

other words, REase and MTase functions can be uncou-

pled, but at the cost of greatly reduced REase activity.

Equally surprising was the effect of the natural cofactor

SAM and its analogue SIN on the REase activity of

TspGWI N473A. SAM, a natural Thermus sp. enzymes

family cofactor, has the least effect on wt TspGWI of all

the members tested to date [16, 17]. In fact, a slight

inhibitory effect was observed with both native (Thermus

sp. GW-isolated) and recombinant wt TspGWI, reflected

by minor differences in the stable partial DNA cleavage

pattern [16] (Fig. 3). Such an inhibitory effect is even more

evident in this work, where non-saturating enzyme con-

centrations are used together with the 390 bp PCR product,

which contains only two TspGWI sites (Fig. 4).This subtle

effect leads to an important conclusion that although the

recombinant wt TspGWI protein apparently retained the

ability to physically bind SAM, the interaction lost its

functionality, so the REase activity could not be stimulated

by the bound cofactor. Extending the reaction time does

not cause SAM to have any further effect (stimulation) on

wt TspGWI [16]. On the other hand, the MTase of the wt

TspGWI is functional. This raises the question of whether

the TspGWI protein has two separate SAM-binding

pockets or whether binding SAM by the MTase moiety

causes conformational changes, sending a signal along the

polypeptide to the REase moiety. It is interesting that the

very closely related TaqII REase from the same subfamily

(but recognizing a different DNA sequence, thus not being

a TspGWI isoschizomer) is very strongly (positively)

affected by SAM [12]. The effect of SIN is similar in the

case of the mutant TspGWI N473A REase. Besides

reporting the ‘minimal’ case of the ‘minimum form of life’

phenomenon, the major novelty of this work lies in the

description of the SIN effect on the TspGWI N473A

enzyme variant—phenotypic mutation suppression result-

ing in the reactivation of the cleavage function. Wt

TspGWI however responds to SIN with a DNA cleavage

specificity change and minor stimulation. We reported

previously that to enhance the effect of SIN-induced

TspGWI specificity relaxation to a 3-bp REase, a molar

excess of the enzyme/recognition site ratio and a prolonged

Fig. 6 TspGWI N473A variant REase DNA cleavage specificity

determination. 300 ng of 497 bp custom prolonged PCR, containing

two convergent 50-ACGGA-30 recognition sites (2 sites variant (?/)),

or having TspGWI recognition sites deleted (0 sites variant) were

digested with 4.16 pmol of recombinant wt TspGWI or TspGWI

N473A variants in buffer T, in the presence or absence of 50 lM SAM

or SIN, for 1 h, at 65 �C. a Recombinant wt TspGWI cleavage pattern

of 497 bp ‘zero’ or ‘2 sites’ variant PCR substrates. Lanes M,

GeneRulerTM 100 bp DNA Ladder (Thermo Fisher Scientific),

supplemented with DNA markers as in Fig. 4 (selected bands

marked); lane K1, untreated 497 bp ‘zero sites’ variant PCR substrate;

lane K2, untreated 497 bp ‘2 sites’ variant PCR substrate, lane 1–3,

‘zero sites’ variant 497 bp PCR DNA cleaved with the wt TspGWI

protein variant, lane 4–6, ‘2 sites’ variant 497 bp PCR DNA cleaved

with the wt TspGWI protein. The reaction products were resolved on

15 % polyacrylamide gel in TBE buffer and stained with Sybr

Green I. b As in Panel a, except that the reactions were carried out

with the TspGWI N473A protein variant

Mol Biol Rep

123

incubation time had to be used [11]. Here, even when SIN

is used under enzyme non-saturating (no enzyme excess)

conditions (precisely like SAM: see Figs. 3, 4; Table 1), it

has a striking effect on DNA cleavage restoration of

TspGWI N473A as compared to the wt TspGWI protein,

though depending on the DNA substrate to a different

extent (Figs. 3, 4). Although SIN changes the specificity of

wt TspGWI, it does not speed up the DNA digestion

reaction (Figs. 3, 4) [11, 16] (this work). Surprisingly,

however, it nearly completely suppresses the N473A

mutation phenotype, resulting in an increase in TspGWI

N473A activity to over 25–50 % of that of wt TspGWI

REase, depending on the substrate used (Fig. 3, 4;

Table 1). The overall stimulation of the TspGWI N473A

mutant by cofactor analogue SIN is determined as a range

of multiplicity rather than a precise number, owing to the

limitations of the REase assay and the gel staining method

used. Nevertheless, SIN-induced TspGWI N473A restric-

tion activity restoration was clearly observed, with an app.

128-fold activity increase compared to the reaction without

SIN supplementation (Fig. 3d–f), and with multiple T7

substrate DNA recognition sites. Under these conditions

(the presence of SIN, T7 substrate DNA), moreover,

TspGWI N473A REase exhibited app. 50 % of the activity

of the wt TspGWI enzyme. In the case of the 390 bp DNA

substrate the stimulation factor could not be calculated, as

there was no DNA cleavage by TspGWI N473A in the

absence of SIN (Fig. 4d). The addition of SIN resulted in

the appearance of the expected DNA digestion products

(Fig. 4f, lane 1), with an intensity corresponding to the

partial cleavage pattern observed for wt TspGWI (Fig. 4a,

lane 3).The relative specific activity of TspGWI N473A

REase for the PCR fragment (two convergent recognition

sequences) in the presence of SIN was therefore estimated

at app. 25 % of wt TspGWI REase activity. The differ-

ences observed for T7 DNA and PCR product substrates

could be attributed to the linear diffusion factor effect, as

T7 DNA is app. 100 times longer than the PCR product and

contains multiple TspGWI sites. To our knowledge, this is

the first example of cofactor analogue-induced mutant

REase activity restoration among REases and possibly

among other SAM-utilizing enzymes. We hypothesize that

the TspGWI N473A mutant has a more relaxed tertiary

structure, with DNA cleavage catalytic aa residues shifted

away from optimal positions. The SIN effect would act as a

‘molecular staple’, physically stabilizing aa residues within

a SAM-binding pocket, thus restoring its competence to

send conformational signals along the TspGWI N473A

mutant polypeptide to the DNA scission catalytic centre,

which has not been directly damaged by a distant mutation

in the tspGWIRM gene section, coding for the NPPY

methylation motif. The experiment shown in Fig. 5 indi-

rectly confirms this conclusion. Both the wt TspGWI and

TspGWI N473A mutant were used in digestions under

titrated down concentrations of SIN. Substantial differ-

ences have been observed: wt TspGWI activity only

slightly decreases over a very wide SIN concentration

range (375 nM–100 lM), while TspGWI N473A restored

activity drops dramatically much sooner (below 0.1 lM).

Comparison of the digestion extent in Fig. 5a and Fig. 5b

shows approximately the same partial digestion in lane 10

of Fig. 5b (TspGWI N473A) as in the lowest SIN concen-

tration tested for wt TspGWI (Fig. 5a, lane 19). However,

this comparison is affected by the wt TspGWI capability to

digest DNA in the absence of SIN, with a 5-bp cognate

recognition site specificity [15]. In the presence of SIN, wt

TspGWI cuts DNA with a mixed specificity, e.g. 5-bp 50-ACGGA-30 [15] and 3-bp SIN-generated ‘affinity star’

specificity [11]. Thus, TspGWI N473A variant is a good

model for ‘isolated’ analysis of the cofactor analogue effect

on the REase, proving the crucial role of SIN binding in

stabilizing cleavage-competent TspGWI N473A molecules.

These experimental data may lead to a further conclusion

that in ‘real life’ (no SIN preset) cellular environment,

SAM stimulation of the undamaged Thermus sp. family wt

enzymes is similar to that of TspGWI N473A by SIN. As

this cofactor analogue is not a methyl group donor and it is

anticipated that it has no affinity for the DNA cleavage

motif PD-(D/E)XK, the interaction is allosteric, acting

from a distant SAM(SIN) binding motif. The experiments

shown in Figs. 3, 4, 5 shed some light on both activity

modulation and specificity transition (wt TspGWI) and

activity reactivation (TspGWI N473A) mechanisms.

Because at the lowest activating concentrations of SIN,

only a fraction of TspGWI N473A variant molecules

(probably wt TspGWI as well, but the presence of the 5-bp

cognate specificity complicates experimental interpreta-

tion) would form a complex with SIN (Fig. 5b, lanes

12–14), the possible activation scenario would need to

include rapid diffusion of a relatively small SIN molecule

away from the labile enzymes-SIN complexes in two

putative modes: (i) from DNA bound TspGWI

N473A(TspGWI)—SIN complexes, while remaining DNA-

TspGWI N473A(TspGWI) complexes would retain a

cleavage activation state or (ii) the transiently SIN-com-

plexed TspGWI N473A(TspGWI) proteins would reach

activation state while in a solution and remain activated

after SIN dissociation, until DNA cleavage occurs. Both

scenarios assume a multi turnover ‘catalytic’ action of

SIN(SAM) on TspGWI enzymes, changing their polypep-

tide folding status. The discussion above did not account

for a chemical binding constant value, as it has not been

determined. Nevertheless, the binding constant rules that

even at stoichiometric concentration of SIN and the

TspGWI variants, not all of them will form complexes,

further reinforcing the conclusions drawn above,

Mol Biol Rep

123

concerning the need for SIN to transiently bind and activate

the protein molecules. Alternatively, one can explain the

sub-stoichiometric stimulation by the presence of mostly

inactive protein molecules within the TspGWI variants

preparations used. Nevertheless, the isolation protocols

used gentle purification methods and the proteins are

thermostable, thus we expect their high stability. During

storage of the purified preparation for over a year at

-20 �C, no activity losses were observed (not shown).

The reactivation phenomenon also raises the question

whether the restored DNA cleavage activity of the TspGWI

N473A mutant remains the same as the wt TspGWI scission

specificity. To address this problem we have generated two

PCR substrates of 497 bp, based on an extension of the

390 bp substrate, evaluated in Fig. 4. The substrate

extension has been done with longer primers for the pur-

pose of precise determination of a digestion product’s

length using polyacrylamide gel electrophoresis. Compar-

ative digestion of a 497 bp substrate with two 50-ACGGA-

30 cognate sites and a 497 bp substrate with no TspGWI

recognition sites has shown that: (i) the cleavage pattern of

wt TspGWI and TspGWI N473A is the same and (ii) the

control substrate without 50-ACGGA-30 cognate sites is not

digested by neither the wt TsoI nor TspGWI N473A mutant

(Fig. 6). Taken together, these digestions show that reac-

tivated TspGWI N473A mutant specificity remains the same

as wt TspGWI.

In the experiments described in this publication, a partial

DNA cleavage (a frequent feature of other subtype IIC/IIG

enzymes) hindered the quantitative evaluation of the REase

activity, in addition to the somewhat subjective nature of

the REases activity assay. For comparing specific activities

we decided to use relative values (obtained in a series of

experiments, exemplified in Fig. 3a–f) rather than the

classic specific activity units used for completely cleaving

REases, owing to the partially subjective nature of REase

unit estimation and the inability of Thermus sp. family

enzymes to cleave substrate DNA completely. Series of

photographs of gels segments as well as single DNA bands

were scanned using UN-SCAN IT GEL software, taken

with various exposure times and cross-calibrated baselines/

intensities. The black arrows in Fig. 3 and Fig. 4 indicate

the lanes of estimated identical or nearly identical extent of

DNA digestion. The red arrows in Fig. 3 indicate the

minimum amounts of the enzyme sufficient to obtain a

stable partial digestion pattern. Despite a certain error and

quantitative analysis limitations, Figs. 3, 4, 5 and 6

undoubtedly show the novel DNA cleavage activity reac-

tivation phenomenon described. We believe that these

findings, besides extending basic knowledge of REase-

cofactor-DNA interaction, will also serve as a more general

method for the alterations of specificities, activities and

fidelities of IIS/IIC/IIG enzymes. Targeting a SAM-bind-

ing pocket or catalytic motifs of MTase with site-directed

mutagenesis, followed by investigation of SAM and its

analogues influence on the activity of the obtained enzyme

variants, may bring novel, artificial REase prototype

specificities not existing in nature or improve the cleavage

characteristics of existing enzymes for DNA manipulation

purposes. Moreover, the method could be used for the

functional conversion of a substantial number of prototypes

already found, which, however, are not used in gene

cloning methodology because of their low Fidelity Index

[12, 25] or partial cleavage feature.

Conclusions

(i) The TspGWI N473A protein variant with altered

methylation of the NPPY motif was expressed and

isolated. Under standard reaction conditions it exhibits

no MTase or trace REase activities (less than 0.8 % of

DNA cleavage of wt TspGWI).

(ii) SIN—the SAM analogue with an inverted charge—

stimulates impaired TspGWI N473A mutant and

reactivates its cleavage function, at a level of

25–50 % activity of the wt protein.

(iii) The SIN-mediated phenotypic mutation suppression

effect is the first described to date. It indicates that

even though the REase and MTase functions reside

Table 1 Relative specific activities comparison of wt TspGWI and TspGWI N473A variant in the presence or absence of SAM or SIN

Relative specific activity of DNA cleavage (%)

Bacteriophage T7 DNA (90 recognition sequences per

DNA molecule) initial molar ratio of enzyme to recognition

site 4.2:1

390 bp PCR fragment (2 convergent recognition

sequences per DNA molecule) initial molar ratio of

enzyme to recognition site 1.82:1

REase Without cofactor SAM SIN Without cofactor SAM SIN

wt TspGWI (recombinant) 100 % 100 % 100 % 100 % 12.5–25 % 100 %

TspGWI APPY

(N473A) mutant

Trace activity

(less than 0.8 %)

Trace activity

(less than 0.8 %)

50 % No activity No activity 25 %

Mol Biol Rep

123

in distant portions of the TspGWI polypeptide,

intense interdomain communication is still taking

place.

(iv) The TspGWI N473A mutant clone exercises a strong

selection pressure on a recombinant E. coli host,

causing long filaments formation and apparently

following, further minimized, Kobayashi’s ‘mini-

mum form of life’ hypothesis [23, 24].

(v) The method for changing the characteristics of IIS/

IIC/IIG enzyme is more general and may serve as a

tool for DNA recognition specificity and fidelity

engineering.

Acknowledgments The authors would like to thank Janusz Bujnicki

for predicting the functionally important amino acid residues of

TspGWI protein, Katarzyna Sliwinska for her valuable technical

assistance and Marta Skowron for editorial corrections. This work

was supported by the Faculty of Chemistry, University of Gdansk

BMN 538-8171-B012-13 and DS/530-8171-D388-13.

Open Access This article is distributed under the terms of the

Creative Commons Attribution License which permits any use, dis-

tribution, and reproduction in any medium, provided the original

author(s) and the source are credited.

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